Compositions and Methods for Assessing Gastrointestinal Health

ABSTRACT

The present invention relates to kits designed for the collection of stool samples and methods of analyzing those samples for biological markers of maldigestion, inflammation, and imbalanced gut flora.

FIELD OF THE INVENTION

The present invention relates to kits designed for the collection ofstool samples and methods of analyzing those samples for biologicalmarkers of maldigestion, inflammation, and imbalanced gut flora.

BACKGROUND

Irritable bowel syndrome (IBS) is a functional disorder of the intestinecharacterized by altered bowel function (diarrhea, constipation, orboth) and abdominal pain. It is the most common gastrointestinaldisorder seen in general practice, and the prevalence of IBS has beenestimated to be as high as 20% of the general population of NorthAmerica. The majority of IBS sufferers are women, and women account for80% of the cases of severe IBS.

SUMMARY

The methods described herein offer a concise look at the overall healthof a patient's gastrointestinal (GI) tract. They include a number ofnon-invasive tests that evaluate digestion, inflammation, and gut flora.The outcome of these tests can assist in the diagnosis and treatment ofIBS. Accordingly, in one aspect, the invention features methods ofassessing a patient's gastrointestinal health by providing a stoolsample from the patient and subjecting the sample to three tests: afirst test for maldigestion, a second test for inflammation, and a thirdtest for gut flora. Testing for maldigestion can include assessments ofdigestion and/or absorption of nutrients from the GI tract. For example,the first test for maldigestion can include testing for pancreaticinsufficiency by evaluating the amount (e.g., the concentration) of apancreatic elastase (PE), or a heterologous protein that is co-regulatedwith PE, in the stool sample. The PE can be the isozyme pancreaticelastase 1 (PE1). Concentrations of PE1 that are more than about 200 μgper gram of stool, but less than about 350 μg per gram of stool,indicate mild pancreatic insufficiency; concentrations of more thanabout 100 μg of PE1 per gram of stool, but less than about 200 μg of PE1 per gram of stool, indicate moderate pancreatic insufficiency; andconcentrations of less than about 100 μg of PE1 per gram of stoolindicate severe pancreatic insufficiency. When desired (e.g., whenpancreatic insufficiency is moderate to severe), the methods can alsoinclude a step of testing for intestinal permeability, generating acomprehensive parasitology profile, and/or testing for Celiac disease.When desired (e.g., when pancreatic insufficiency is moderate tosevere), the methods can also include a step of assessing boneresorption, analyzing glucose and/or insulin levels, testing for Celiacdisease, and/or testing for bacterial overgrowth in the small intestine.

The second test, for inflammation, can be carried out by testing forcalprotectin or occult blood in the stool sample. The testing forcalprotectin can be carried out by evaluating the amount of calprotectin(e.g. its concentration), or a heterologous protein that is co-regulatedwith calprotectin, in the stool sample. Calprotectin elevated to about50-120 μg per gram of stool indicates low-grade inflammation of thegastrointestinal tract; calprotectin elevated to more than about 120 μgper gram of stool indicates significant inflammation in the GI tract;and calprotectin elevated to more than about 250 μg per gram of stoolindicates severe inflammation in the GI tract. Elevation to over about250 μg/g also indicates that a patient with inflammatory bowel diseaseis at high risk of relapse within one year. Signs of inflammation can beanalyzed further by assessing intestinal permeability, testing for foodallergies, and/or performing a comprehensive parasitology profile.

The third test, to examine gut flora, includes testing for harmful andimbalanced gut flora and preferably includes testing for both harmfuland non-harmful (e.g., non-pathogenic or beneficial) gut flora. Harmfulgut flora includes, but is not limited to, Clostridium difficile andHelicobactor pylori; imbalanced gut flora includes, but is not limitedto, Klebsiella species, Pseudomonas species, and Clostridial species;and beneficial gut flora includes, but is not limited to, lactobacillusspecies, bifidobacteria species, and Escherichia coli. Where there is apresence of harmful bacteria, an excess of imbalanced gut flora and/orinsufficient beneficial gut flora, there is a maldistribution of florawithin the total community. The presence of harmful bacteria, an excessof imbalanced flora and/or an insufficiency of beneficial gut flora areall forms of dysbiosis. Treatment for dysbiosis can include probiotictreatment, and when the gut contains harmful gut flora or imbalancedflora, a patient can also be treated with anti-microbial herbs and/orantibiotics.

Regardless of outcome, the methods of the invention or a step within themethods (e.g., testing for inflammation) can be repeated at a laterpoint in time (e.g., six weeks later). Thus, the methods of theinvention can be repeated periodically and used to monitor a patient.The monitoring can determine whether a treatment (e.g., a drugtreatment) or lifestyle change (e.g., a change in diet or exercise) ishaving a measurable effect on the tested parameter(s).

In another aspect, the invention features kits that can be used toprovide stool specimens in a condition suitable for testing in themanner described above. For example, a kit for the collection of a stoolsample can include (a) a collection tub; (b) a tube containing afixative medium; (c) a tube containing a medium that maintains therelative proportions of organisms in a stool sample; and (d) writtenmaterials. The fixative medium maintains the integrity of organismswithin the stool for analysis, and a suitable example is SAF medium. Themedium that maintains the organisms (i.e., a medium that does notselective kill or selectively support any given organism) can beCary-Blair medium.

The written materials can be presented in various forms and can includeone or more of: (a) instructions for use; (b) a requisition form; and(c) a mailing envelope or other materials for transporting the sample.

In addition to the components listed above, the kit can include otheritems such as one or more of: (a) a holder to suspend the collection tubover a toilet; (b) an empty cup; (c) an absorbent pad; (d) a flat toolsuitable for insertion into a hand-held tube (e.g., a wooden stick suchas a tongue depressor or a similarly shaped item made from wood,plastic, or other materials); and (e) a glove (e.g., a disposable glove,which may be biodegradable).

An IBS diagnosis is based on identifying positive symptoms consistentwith the condition and excluding other conditions with similar clinicalpresentations. IBS symptoms often mimic those associated with other GIconditions, such as maldigestion and disorders of absorption (e.g.,celiac disease, lactose intolerance, pancreatic insufficiency),infection and dysbiosis, as well as inflammatory bowel disease. IBS isdifferentiated from IBD (irritable bowel disease) in that, unlike IBD,IBS does not cause severe inflammation, ulcers or other damage to thebowel. Where the only diagnosis is a diagnosis of exclusion, the averagetime from the onset of symptoms to a positive diagnosis of IBS is nearlythree years. In addition, incorrect symptom attribution may lead toreferral to a gastroenterologist and unnecessary procedures (such ascolonoscopy or endoscopy), hospitalization, or surgery (e.g.,appendectomy, cholecystectomy, or hysterectomy). Currently, Rome IIIcriteria are used to diagnose functional gastrointestinal disorders suchas IBS, although these criteria are not significantly utilized inprimary care (as they do not differentiate therapeutic choices).Diagnosis based on tests to evaluate digestion, inflammation, andinfection/gut microflora will result in more timely and accuratediagnosis of IBS.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a culture plate, divided intoquadrants, that has been inoculated and streaked using the spreadingpattern shown.

FIG. 2 is a flow chart depicting representative analytes and pathogensthat can be assessed to evaluate digestion, inflammation, and gut florain a patient.

FIG. 3 is a representative report illustrating the manner in whichinformation obtained in the methods of the invention can be conveyed toa clinician. The report can be supplied physically or by computergraphics.

DETAILED DESCRIPTION

The compositions and methods described herein can be used to assessgastrointestinal health in virtually any patient, regardless of age andregardless of whether or not the patient has any specific symptom orcomplaint (i.e., the method can be carried out on a patient in goodhealth). In many cases, however, the patient will be a person who iscomplaining of abdominal pain, perhaps associated with gas or bloating,constipation or diarrhea, or other symptoms of a gastrointestinaldisease or disorder.

Chronic maldigestion can lead to bacterial or fungal overgrowth andalterations in gut permeability. Toxins and large molecules that escapethe intestinal barrier can enter the general circulation, inflame theliver, burden the body's detoxification system, and increase the riskfor food allergies, joint disease, and imbalances in overall health.Malabsorption can lead to deficiencies of nutrients, proteins,carbohydrates and fats. This can result in long term healthcomplications such as anemia, malnutrition, impaired metabolism andother diseases, such as osteoporosis. Chronic dysbiosis (a microbialimbalance) can lower the levels of beneficial short chain fatty acidsand alter bacterial metabolic activity, thereby increasing the risk ofcarcinogenesis, hormonal imbalance, and GI inflammation. Altered GIimmune function and exposure to bacterial pathogens can lead todiarrhea, mucosal inflammation, intestinal permeability, toxinproduction and autoimmune disorders. Although the invention is not solimited, patients suspected of suffering from one or more of theseconditions (e.g., patients complaining of any of these symptoms orexhibiting signs of any of these conditions) are candidates for testingas described herein. Further, any of the methods described herein caninclude a step of identifying a patient as a candidate for testing(e.g., a patient complaining of gastrointestinal distress or exhibitingsigns of an associated condition). For example, the methods can includea step of questioning a patient about their gastrointestinal healthand/or performing a physical examination.

Digestion can be assessed by testing for pancreatic sufficiency orinsufficiency, as the case may be, which can, in turn, be assessed bydetermining the level of expression or activity of a protein that isproduced primarily or exclusively by the pancreas and that remainsstable or detectable following passage through the intestine. Forexample, one can assess a pancreatic elastase (PE), for example PE1, oranother marker of pancreatic function (e.g., a protein that isco-regulated with pancreatic elastase). Pancreatic elastases areproduced by the human pancreas and, as noted, are an indicator ofexocrine pancreatic function, as levels of PE are largely unaffected bytransit through the GI tract or enzyme supplementation. These levelscorrelate with duodenal outputs of amylase, lipase, trypsin, andchymotrypsin, which may also be assessed. PE levels from 100-200 μg/g ofstool are associated with moderate pancreatic insufficiency, whereasvalues below 100 μg/g indicate severe pancreatic insufficiency.Clinically, decreased levels of PE in stool reflect the need forexogenous digestive enzyme supplementation to support exocrinepancreatic deficiency.

Expression and activity of PE can be assessed in numerous ways usingtechniques known in the art. For example, PE expression can be detectedwith proteins that specifically bind PE (e.g., an anti-PE antibody). Ina particular embodiment, PE expression can be assessed using acommercially available test, such as the ScheBo® PE1 kit (ScheBoBiotech, Giessen Germany). A random stool sample can be collectedaccording to the patient instructions provided with the kit. Briefly, a100 ml polypropylene container is filled ¼-⅓ full with stool by thepatient and homogenized using the tool (e.g., a wooden tongue depressor)provided. The container is closed tightly, placed in a shipping device,and sent to the laboratory. Stool samples should be received by the labwithin 8 days of collection and are stable for three days thereafterwhen stored at 2-8° C. Frozen aliquots are set up in 1.5 mlmicrocentrifuge tubes for longer storage at −20° C. Of course, samplesmay be provided otherwise. For example, samples may be collected fromhospitalized patients and taken directly to the hospital's lab or anearby testing facility.

In the ScheBo® PE assay, an ELISA plate is coated with a monoclonalantibody that specifically binds human PE1. PE1 present in a sample orstandard is bound to the antibody and thereby immobilized on the plate.A complex of monoclonal anti-PE1-biotin and peroxidase(POD)-streptavidin binds to PE during the next incubation. Theperoxidase oxidizes ABTS(2,2′-azino-bis(3-ethylbenzothiazolin-6-sulfonic acid)diammonium salt),which turns dark green. Finally, the concentration of oxidized ABTS isdetermined photometrically. Polyclonal antibodies can also be used todetect proteins such as PE1 that are produced by the pancreas and stablytransported through the gastrointestinal tract.

As noted, PE1 serves to indicate pancreatic function (e.g., sufficiencyor insufficiency). Where levels are greater than about 200 μg/g ofstool, exocrine pancreatic function is adequate and no further action isnecessary with respect to pancreatic function. However, a physician maywish to consider pancreatic supplementation if levels are in the lownormal range (e.g., 200-350 μg/g). Healthy individual produce more than500 μg/g of PE. Thus, levels between about 200 and 500 μg/g suggest adeviation from optimal pancreatic function. Where this outcome is found,the clinician should consider digestive enzyme supplementation if one ormore of the following conditions is present: loose, watery stools;undigested food in the stool; post-prandial abdominal pain; nausea orcolicky abdominal pain; gastroesophageal reflux symptoms; bloating orfood intolerance. Low levels of PE1 (for example, ˜100-200 μg PE/g ofstool) indicate mild to moderate pancreatic insufficiency and pancreaticenzyme supplementation as well as further testing to assess intestinalpermeability and to determine the profile of intestinal parasites isadvised. Celiac testing can also be performed. Very low levels of PE1(for example, <˜100 μg PE per gram of stool) indicate moderate to severepancreatic insufficiency. In this event, pancreatic enzymesupplementation and vitamin and mineral supplementation are indicated.

In addition to treatments targeted to the pancreas, physicians may wishto consider other medical tests and treatments. For example, reduced PEis found in over 50% of type 1 diabetics and 35% of type 2 diabetics.Diabetes secondary to exocrine disease could be much more frequent thanpreviously thought; studies have shown that low PE is closely related toglycemic control. Exocrine pancreatic function is also frequentlyimpaired in gallstone sufferers and post-cholecystectomy patients. Thereis a high prevalence of phathological changes in exocrine pancreaticfunction in patients with gallstones. Nearly one third of patients withosteoporosis have reduced concentrations of PE. Vitamin D levels mayalso be significantly decreased in these patients. PE is also useful inmonitoring exocrine pancreatic function caused by: chronic pancreatitis,auto-immunopathies and connective tissue diseases, chronic inflammatorybowel disease, and intestinal malabsorption with mucosal atrophy. Atransient reduction of PE can occur with villous atrophy. After mucosalregeneration, PE levels return to normal. PE can therefore be used as amonitoring tool in patients with Celiac disease and other malabsorptiveconditions. Accordingly, the present methods in which digestion isassessed by examining PE can be carried out periodically to monitor apatient's condition and/or recovery, and the methods can be expanded toinclude other tests such as those mentioned above. For example, furthertesting can include one or more of: bone resorption assessment,glucose/insulin analysis, Celiac testing, and testing for bacterialovergrowth in the small intestine.

Another way to assess digestion is by evaluating stool alpha 1chymotrypsin levels or assessing fecal fat content. Chymotrypsin is anenzyme secreted by the pancreas that functions in protein digestion.Levels of stool chymotrypsin can be determined using a stool sample thatis collected as described herein and preserved in a minimal microbialgrowth media. A high salt surfactant can be used to release thechymotrypsin contained within the sample. The reaction of the freechymotrypsin with a spectrophotometrically active substrate facilitatesthe determination of chymotrypsin activity in the sample. Results can beexpressed as units of chymotryptic activity relative to the grams ofstool analyzed. A normal result for this assay is ˜0.9-26.8 U/g stool.Low levels of chymotrypsin (<0.9 U/g) indicate exocrine pancreaticinsufficiency. Therapy should include exogenous supplementation ofpancreatic enzymes including lipase. Elevated levels of chymotrypsin(>26.8 U/g) suggest a rapid transit time (diarrhea). A faster transittime reduces the intestinal degradation of chymotrypsin, which resultsin an increased recovery of this enzyme. Chymotrypsin could also beelevated with excess pancreatic enzyme supplementation.

As both pancreatic elastase and chymotrypsin are indicators of exocrinepancreatic function, the clinical indications for PE also apply tocymotrypsin. Therefore, the methods in which chymotrypsin is assessedcan also include a step of identifying a patient who has: loose, waterystools, undigested food in the stool, post-prandial abdominal pain,nausea or colicky abdominal pain, gastroesophageal reflux symptoms, orbloating or food intolerance. Other conditions associated with reducedchymotrypsin include diabetes, cystic fibrosis, chronic pancreatitis,and malabsorption.

Both chymotrypsin and PE are highly accurate in distinguishing betweenpancreatic maldigestion and intestinal malabsorption (82% and 92%,respectively). PE is not affected by bovine or porcine enzymesupplements, so patients do not have to discontinue therapy to assessbaseline levels. Chymotrypsin is affected by exogenous supplementation,which makes it an ideal tool to monitor dosing adequacy. Whenchymotrypsin values fall within the reference range in supplementedindividuals, the clinician can be confident that an appropriate dosageof digestive enzymes is being administered. As noted, the presentmethods can be carried out not only in the context of an initialpresentation and diagnosis, but also in the context of ongoingmonitoring. While PE may be a more accurate non-invasive marker toassess exocrine pancreatic function, chymotrypsin is the preferredmarker to monitor enzyme supplementation.

Elevated levels of fecal fat (e.g., elevated cumulative levels) are alsoan indicator of maldigestion. Fecal fats include triglycerides, longchain fatty acids (LCFAs), cholesterol and phospholipids, which can beextracted with methods known in the art and measured individually toobtain a measure of fecal fat levels. To evaluate fecal fat, a stoolsample can be submitted in a preservative (e.g., 5% formalin), andlipids can be extracted from the sample using an organic extraction.Once the stool lipids are isolated, standard automated chemistry assayscan be used to determine the levels of the individual fecal fatcomponents, including triglycerides, cholesterol, phospholipids and longchain fatty acids. Cumulative totals of these components outside therange of 2.6 to 332.4 mg/g stool are indicative of malabsorptionTriglycerides represent the major component of dietary fat (on average120 g of a 125 g daily load). Elevated levels are suggestive ofincomplete fat hydrolysis, which can be caused by exocrine pancreaticinsufficiency or bile acid insufficiency. Elevated triglycerides withnormal LCFAs have been noted in patients with steatorrhea due topancreatic insufficiency. Triglycerides may also be elevated with rapidtransit time, which impairs the breakdown and absorption of theselipids.

LCFAs are normally readily absorbed in a healthy mucosa. Elevated levelssuggest malabsorption, reduced pancreatic function or bileinsufficiency. Increased LCFAs have also been noted after acuteintestinal infections.

Fecal cholesterol is derived from the diet, bile, and from mucosalepithelial break-down. In a healthy gastrointestinal tract, about 40-60%of dietary cholesterol will be absorbed. Elevated levels are areflection of mucosal malabsorption. Impaired absorption of fecalcholesterol occurs in Celiac disease secondary to damaged mucosa fromgluten ingestion.

Phospholipids are derived from three specific sources: bile (50%), diet(25%), and mucosal desquamation (25%). The major dietary-derivedphospholipids include phosphatidyl choline, phosphatidyl serine,phosphatidyl ethanolamine and cardiolipin. In a healthy individual,nearly 85% of intestinal phsopholipids are absorbed. As phospholipidsare derived from more than one source, elevations could occur from thefollowing: malabsorption, inadequate bile salt resorption, or increasedmucosal cell turnover. Referring to other absorptive markers can helpdetermine the cause of high fecal phospholipids.

The following conditions can contribute to the impaired absorption offecal fat: pancreatic insufficiency (specifically lipase), cholestasis(e.g., bilary obstruction or liver disease), interrupted enterohepaticcirculation (e.g., ileal disease or bile sale deconjugation from smallbowel bacterial overgrowth), Celiac disease, short bowel syndrome, andWhipple's disease (rare). In general, an elevation in any one of thefecal fat markers is suggestive of malabsorption (see above for theclinical significance of individual absorptive markers).

Inflammation affecting the GI tract can be detected in a variety ofways, including by tests for calprotectin expression and occult blood.Calprotectin is a calcium-binding protein found primarily in neutrophilsthat serves as a direct marker of intestinal inflammation. Infection,tissue damage, or increased permeability of the mucosa results in themigration of granulocytes into the intestinal lumen. As with PE,calprotectin can be detected by methods known in the art, includingmethods in which a calprotectin-specific binder is used to detectexpression. For example, one can use the PhiCal® quantitative EIA(Calpro AS).

Calprotectin is a useful analyte because it is resistant to bacterialdegradation in the gut and is stable in stool for up to one week at roomtemperature. It is also unaffected by medications, dietary supplements,and enzymatic degradation. Further, calprotectin: reflects the flux ofleukocytes into the intestinal lumen; is released upon activation anddegranulation of neutrophils; correlates strongly with111-indium-labeled leukocyte excretion as well as histologic andendoscopic grading of disease activity in ulcerative colitis; helpsdifferentiate between IBS and active IBD; predicts relapse in patientswith IBD and serves as an objective marker to assist in treatmentprotocols; and assists in selecting patients for endoscopy and inmonitoring responses to treatment (especially in children who mayrequire general anesthesia to undergo more invasive analyses).

For calprotectin analysis, a random stool sample is collected accordingto patient instructions for the calprotectin PhiCal® test. Briefly, asuitable container (e.g., a 100 ml polypropylene tube) is filled ¼-⅓full with stool by the patient and homogenized using a tool, such as awooden tongue depressor. Controls for low and high calprotectinexpression can be assayed with each batch of samples. PhiCal® uses apolyclonal antibody against calprotectin that is absorbed to the surfaceof a plastic well to bind calprotectin in diluted stool samples. As theantibody is linked to an enzyme, a subsequent reaction in which theenzyme catalyzes a reaction to yield a detectable (e.g., fluorescent orcolored) product provides an easy means for detecting and quantitatingbound calprotectin. Values below ˜50 μg of calprotectin per gram ofstool are not indicative of inflammation in the GI tract, and no furtheraction is necessary based on this result. Values between 50-120 μg/gstool are associated with low-grade inflammation, which could be due topost-infectious irritable bowel syndrome (IBS), infection, foodallergies, polyps, neoplasia, non-steroidal anti-inflammatory drugs(NSAIDs) or IBD in remission. It is prudent in these cases to repeat thecalprotectin assay after about six weeks. If levels remain elevatedafter ruling out other etiologies, further investigative tests (e.g.,endoscopy or imaging) should be considered. These further tests include:stool culture, an intestinal permeability assessment, a food antibodyassessment, and a comprehensive parasitology profile. Therapeuticintevention includes probiotics, fish oils, N-acetylglucosamine, and/orrutin. Calprotectin expression above ˜120 μg/g indicates significantinflammation, possibly caused by IBD, infection, food allergies, NSAIDuse, polyps, adenomas, colorectal cancer, or diverticulitis. Unless thesource of the inflammation is clear, further evaluation is recommendedand may include endoscopy and/or colonoscopy. One can also assessmicrobiology/parasitology at this stage, and further testing includesany of the tests listed above in connection with lower elevation ofcalprotectin. Where calprotectin is severely elevated (>250 μg/g), thereis a strong likelihood that the patient has active IBD or are at a highrisk of relapse to active IBD within one year. In this event, IBD shouldbe managed with standard therapies. Intervention can includeadministration of probiotics, fish oils, N-acetylglucosamine, rutin,and/or anti-inflammatory agents (e.g., leukotriene inhibitors andTNF-alpha antagonists). For patients with IBD, calprotectin levelsbetween 250-500 μg/g indicate low to moderate disease activity. Levelsabove 500 μg/g suggest high disease activity. Patients with IBD inremission and levels above 250 μg/g have a high risk of relapse withinone year.

Where calprotectin levels are above 120 μg/g, there is likelysignificant inflammation in the gastrointestinal tract. The cause couldbe IBD, infection, NSAID use, polyps, adenomas, or colorectal cancer.Calprotectin may also be elevated in children with chronic diarrheasecondary to cow's milk allergy or multiple food allergies. Furtherprocedures can be carried out to determine the cause more specifically.Levels ranging from 50-120 μg indicate less severe inflammation. Valuesbelow 50 μg/g indicate there is little or no inflammation in thegastrointestinal tract.

Regardless of the cause, patients with elevated calprotectin should betested further with, for example, an intestinal permeability assessment,an allergy antibody assessment, Celiac panel, ImmunoGenomic™ profile, ora comprehensive parasitology profile.

Another marker for inflammation is an eosinophil protein (e.g.,eosinophil protein X or EPX, also known as eosinophil-derived neurotoxin(EDN)). In healthy individuals, eosinophils reside in the connectivetissue layer of the gut, known as the lamina propria. It is not untildamage occurs to the lamina propria that eosinophils migrate into thegut lumen. Eosinophils contain a number of highly cationic proteins suchas eosinophil cationic protein, major basic protein, eosinophilperoxidase, and EPX. Upon eosinophil degranulation, these cationicproteins are release and can be assayed. As the proteins possesscytotoxic properties, their accumulation is associated with inflammationand tissue damage.

While assessing eosinophilic activity is informative, tests that requirecolonoscopy or biopsy have limited utility for office-basedpractitioners. Eosinophil markers in stool, however, can be assayedaccording to the present methods as indicators of inflammation. As withother markers described herein, eosinophilic markers can provideinformation regarding a patient's initial treatment and can be assayedover time to monitor that treatment. For example, baseline levels of EPXcan be used to determine intestinal inflammation associated with foodallergy and to monitor dietary changes. Studies have demonstrated asignificant reduction in EPX after three months on a successfulelimination diet.

Assays for EPX can be based on extraction of EPX from neat stool using acationic extraction buffer. Once extracted, the protein can be detectedand quantitated using any EPX-specific binding protein (e.g., anantibody). Eosinophils are bone-marrow derived cells that secrete arange of highly toxic granule proteins and other inflammatory mediators.The cells are characterized by the presence of dense granular depositsin their cytoplasm that contain proteins that mediate the inflammatoryresponse and tissue damage. EPX is one of the highly cationic proteinscontained within these granules. Eosinophils play a role in allergy andin the response to parasites and are found in significant numbers in theunderlying connective tissue of the respiratory, gastrointestinal, andurogenital tracts. Biopsies of patients with IBD (ulcerative colitis andCrohn's disease) or Celiac disease have demonstrated marked infiltrationof eosinophils. This infiltration plays a role in the pathogenesis ofthe inflammatory processes associated with these disease states.Further, increased levels of EPX have been demonstrated in the feces ofpatients with active disease.

If desired, EPX can be evaluated using the EDN kit available from theMedical and Biological Laboratories Co., Ltd (MBL). Once extracted, EDXis quantified using a sandwich ELISA. The reportable range of the assayis 0.9 ng/ml to 40.0 ng/ml. Where EPX is present at <˜7.0 μg/g, there isno active inflammation of the GI tract. However, elevated levels of EPX(>˜7.0 μg/g) indicate inflammation and/or tissue damage in the GI tract.Underlying causes include food allergy and/or atopic dermatitis,protein-sensitive enteropathy, helminthic infection, IBD, allergiccolitis, chronic diarrhea, chronic alcoholism, bowel cancer,eosinophilic gasteroenteritis (rare), and gastroesophageal reflux.Increased levels of EPX have also been found in ulcerative colitis andCrohn's Disease, with elevations correlating with disease activity. As anon-invasive marker, EPX offers increased sensitivity for evaluatinginflammatory disease activity and for predicting relapses in patientswith IBD.

Occult blood is blood in the stool that is present in amounts too smallto be seen but large enough to be detected by chemical tests. In ourmethods, we test for occult blood as an indicator of inflammation and asign of certain gastrointestinal disorders. Occult blood can be detectedin several ways, including by immunoassays for hemoglobin. Forconvenience, one can use a commercially available test such as theHemosure® One Step Immunological Fecal Occult Blood (iFOB) test suppliedby Quidel Corporation. This test is a rapid, qualitative, sandwich dyeconjugate immunoassay for the detection of human hemoglobin in feces. Itemploys a unique combination of monoclonal and polyclonal antibodies toselectively identify hemoglobin in test samples with a high degree ofsensitivity. In less than five minutes, elevated levels of humanhemoglobin as low as 0.05 μg hHb/ml can be detected and positive resultsfor high levels of hemoglobin can be seen in the test as early as two tothree minutes. Stool samples in Cary-Blair medium can be tested in thisassay within about seven days, and stools collected in the preservationbuffer tube are stable as follows: for six days at 37° C., six months at4° C., and 20 months at −20° C. A monoclonal antibody-based assay thatcan be used is the Hemosure® test, which is specific for humanhemoglobin.

A positive result can indicate ulcers (e.g., peptic ulcers), polyps,diverticulitis, IBD, or colorectal cancer. Bowel lesions, polyps andcolorectal cancers may not bleed or may bleed intermittently, however,and a test result can be negative even when disease is present for thisreason. Repeated testing is advisable if symptoms persist, andphysicians are likely to order repeated testing for occult blood as wellas other, invasive tests (e.g., colonoscopy) or imaging (e.g., a bariumenema) whenever colorectal cancer is suspected. Patients should becautioned not to take vitamin C supplements for several days prior totesting for occult blood, as ingestion over about 250 mg of vitamin Cper day inactivates the test.

Conversely, a test can be positive in healthy patients because certainmedications may cause gastrointestinal irritation resulting in occultbleeding. These include rectal suppositories and oral medications suchas aspirin and corticosteroids. If a patient has not refrained fromeating undercooked meats, a false positive result may ensue. However,the Hemosure® iFOB test is highly specific and should not react tohemoglobin from fish, beef, chicken, rabbit, or goat. Stool samples mayalso become contaminated with blood if collected during menstrualbleeding or if a patient has blood in the urine. Positive and negativecontrols should be run with each test. The positive control can bediluted human blood, and the negative control can be a known blood-freesample, such as water or a buffered solution.

Accordingly, the present methods can include repeated occult bloodtests, positive and negative controls for occult blood, and a step ofobtaining information from a patient regarding vitamin C intake, meatintake, and menstruation.

Where the present methods assess inflammation, they can also include anassay for lactoferrin. Human lactoferrin is an 80 kilodaltonglycoprotein that binds iron and is secreted by most mucosal membranes.It is a major component of polymorphonuclear neutrophils (PMNs), whichare the primary component of an acute inflammatory response.

Assessing inflammation is especially useful in distinguishing IBS fromIBD, as there is little if any inflammation associated with the formercondition.

Lactoferrin can be assessed in a number of ways, including the IBD-Chek™test, which uses antibodies to human lactoferrin. The microtiter wellscontain immobilized polyclonal antibody against lactoferin. Thedetecting antibody consists of polyclonal antibody conjugated tohorseradish peroxidase. In the assay an aliquot of fecal specimen isemulsified in the diluent and transferred to a microtiter well. Anylactoferrin present in the sample binds to the immobilized antibody.After incubation, the wells are washed and the conjugated antibody isadded. A second wash step removes unbound conjugate. With the additionof substrate, any conjugate present as the result of bound lactoferrinresults in color formation, which can be quatitated with aspectrophotometer. At 450/620 nm, a negative result is <0.160, apositive result is ≧0.160. All positive results should be confirmed byrepeat testing.

In the paragraphs that follow, we discuss methods for assessing gutflora.

Identification of microorganisms is key to determining microbialimbalance due to redistributions within the microbiome, parasiticinfections, and other changes in gut flora. Traditional culture methodsenable the identification and semi-quantitation of specific organismsthrough the utilization of differential growth media. This can also beaccomplished using automated biochemical instruments such as Vitek.Other methods and techniques that can be used include mass spectrometrymethods such as MALDI-TOF-MS (Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry). The methodologyuses species- and strain-specific biomarkers to identify organisms.Microarray technology can also be used to evaluate nucleic acids such as16sRNA, small peptides or molecules such as toxins that are specific forthe microorganism, Carbohydrate (e.g., polysaccharide) profiles can alsobe determined. Nucleic acid probe technology can also be used incombination with fluorescent microscopy for microbial quantification andidentification. Other methods for assessing the microbiome includenucleic acid amplification by PCR and RNA or DNA sequencing.

General stool culture procedure: Methods suitable for use in culturingor otherwise treating stool samples are known in the art and can be usedin the context of the present invention. In the following paragraphs, weprovide information regarding the processing, testing, andinterpretation of stool bacterial cultures. These processes can be usedto generally evaluate the microbial community rather than to diagnoseinfectious disease by analyzing pure cultures of specific organisms.

The microbial community that resides in the gastrointestinal tract iscomprised of numerous species of naturally occurring bacteria. Whilesome defined organisms are clearly harmful, most bacteria that typicallyfound in the GI tract are beneficial and are present in numbers thatpromote good health. More specifically, beneficial bacteria, such aslactobacilli and bifidobacteria, play an important role in promoting ahealthy gut microflora environment and ensuring proper digestion. Theseorganisms can prevent the over-colonization of the gut with pathogenicorganisms and may reduce the risk of certain gastrointestinal diseases.The role of some commensal organisms may vary depending on theirpresence as a percentage of the total bacterial population. Using thepresent methods, one can evaluate the bacteria present in a stool samplein a more comprehensive way. Identifying each organism and enumeratingit relative to other isolates provides the clinician with a picture ofthe microbial community present in the GI tract and, more specifically,the colonic environment, at the time the sample was collected. Thisinformation is useful in determining whether the patient is sufferingfrom clinical gastritis or subtle, atypical aberrations that mayindicate a sub-clinical disease process. The bacteria present can becategorized as harmful (or pathogenic), potentially harmful (orpathogenic), or beneficial.

Several factors may affect the composition of the colonic flora,including diet, transit time, stool pH, age, microbial interactions,colonic availability of nutrients, bile acids, and sulfate, as well asthe ability of the microbes to metabolize these substrates.

The predominant beneficial flora in the large intestine are thebifidobacteria, which constitute as much as 25% of the overall colonicflora in healthy adults. Recovery of these organisms from the colonshould therefore ideally be in the 3+ or 4+ ranges. In the colon,obligate anaerobes such as bifidobacteria predominate over facultativeanaerobes such as lactobacilli by 1000:1. It is for this reason thatlactobacilli growth as low as 1+ or 2+ is considered normal in healthyadults. Non-pathogenic E. coli populate the distal colon, although theyare usually found in reduced quantities, comparable to levels oflactobacilli. A 1+ to 2+ concentration of non-pathogenic E. coli istherefore considered normal.

Organisms of the genera Salmonella, Campylobacter, and Shigella havebeen associated with the production of a broad spectrum of clinicalsymptoms observed in the event of bacterial gastritis. Accordingly,these organisms are designated as pathogens and their presence normallyresults in acute diarrhea. Other traditionally recognized intestinalpathogens include enterotoxigenic E. coli, Shigella, Yersinia,Pleisiomonas, Vibrios, Aeromonas, Campylobacter, the viral pathogenrotavirus, and the parasites Cryptosporidium and Coccidia. A number ofother bacteria have been associated with gastrointestinal discomfort,but their etiologic role is still largely undetermined.

Organisms such as Citrobacter freundii, Enterobacter cloacae, andKlebsiella pneumoniae are usually classified as “normal” flora. However,some studies have associated these and other organisms with variousgastrointestinal complaints when they are the predominant organismsidentified on stool culture in the absence of other frank pathogens. Thepresence of traditionally non-pathogenic bacteria in predominatingnumbers could indicate a dysbiotic state in the colon. These organismsmay be the direct cause of a gastrointestinal disturbance, they may beaggravating such a disturbance, or they may simply be present as anindicator of some other disruptive process. Accordingly, we tend toclassify these organisms as potential pathogens. Yeast have also beenassociated with clinical syndromes related to dysbiotic colonicenvironments. There are no definitively recognized pathogenic yeast, andwe therefore refer to yeast as a potential pathogen when present inelevated numbers. Candida albicans is the most significant isolate ofyeast, and its presence is associated with a broad spectrum of clinicalconditions.

Beneficial bacteria are organisms whose presence in substantial numbershas been associated with a healthy colonic environment. These bacteriainclude Bifidobacterium sp., Lactobacillus sp. and Escherichia coli.

Useful materials include: stool transport (Cary-Blair media); blood agarplates; Maconkey agar plates; colistin-nalidixic acid (CNA) plates;hektoen-enteric agar (HE) plates; oxyrase bidifobacter agar plates;candida ID agar plates; sterile swabs; gloves; isoplater instrument (forautomated streaking); and an incubator.

Samples arriving for testing may be in a transport media that preservesstool bacteria. Preferably, the transport media suspends activemetabolism but preserves viability. A suitable medium is the Cary-Blairformula manufactured by the MML company. Samples preserved in thismedium can be reliably tested up to six days after they have beencollected from a patient.

To inoculate plates, the sample can be mixed before using a sterile swabto transfer a sample of stool onto the surface of a culture plate (e.g.,to about a 0.5×0.5 cm patch). To assess bacteria, one can inoculate oneor more plates holding the following culture media: blood agar (BAP),Hektoen-enteric agar, Maconkey (MAC) agar, colistin-nalidix acid (CNA),candida ID agar, and MCA bifidobacter agar. The sample can then befurther distributed on the surface of the agar either manually (e.g.using a sterile loop or needle) or by an automated streaker instrument.Typically, the MCA bifidobacter agar and the candida ID agar aremanually streaked. The plates are then incubated (e.g., at 30-37° C.)for a number of hours (e.g., 8-24 hours or more) prior to evaluation.Some plates (e.g., the bifidobacter agar plates can be incubated longer(e.g., at 35° C. for at least or about 72 hours). Candida ID agar platescan be incubated for at least or about 72 hours at 35° C.

Following incubation, the plates can be assessed in numerous ways. Forexample, one can begin by noting changes in morphology. The CNA platecan be evaluated for alpha-hemolysis (green), gamma-hemolysis (nohemolysis) and/or beta-hemolysis (clearing of agar immediatelysurrounding a colony). Isolates can be recovered from this plate andothers. Lactobacillus, Streptococcus, and Staphylococcus are a few ofthe isolates that may be recovered from this plate. The HE agar platecan be examined for the presence of lactose (yellow) and non-lactosefermenters, hydrogen sulfide producers (black pigment) and clearlymucoid colonies. These plates are useful in isolating Salmonella, whichproduce hydrogen sulfide, and Shigella, which do not ferment lactose andappear as clear colonies. Maconkey agar can be used to identifygram-negative organisms. Almost all enteric bacilli will grow on thismedia. Lactose fermenting colonies (pink), non-lactose fermentingcolonies (grayish or colorless), and mucoid colonies may appear on theseplates. The blood agar plate can be compared to the other media.Swarming or beta-hemolysis that is present here but not on other platesshould be pursued.

In addition to gross observations regarding morphology, one can identifyand quantitate each organism. Growth quantitation is important fordetermining the significance of the organism. Referring to FIG. 1,organisms can be categorized according to the extent of their growth.For example, organisms growing in the first quadrant only can bedesignated “1+”. Growth in the first and second quadrants would becategorized as “2+”, and so forth. These determinations may be somewhatsubjective. For example, if organisms on the plate are clearly isolated,and there is one discernable colony in quadrant 1 and 1 discernablecolony in quadrant 2, the quantitative report could be very few coloniesor 1+ growth. However, if the reader is unable to determine the amountpresent in quadrant 1 (due, for example, to heavy growth of additionalorganisms), the outcome may be 2+ instead.

Affirmative identification of an organism can be carried out in severalways. Procedures for many different biochemical and media-baseddifferential tests are known in the art. It is generally true that,regardless which test is used, the most accurate identifications aremade when working with a pure culture. Thus, we stress the advantages ofworking on a single, well-isolated colony.

After quantifying and identifying the pathogens, one can assess minimuminhibitory concentrations or perform botanical assays. When indicated(e.g., when requested by a patient's physician), sensitivities andbotanical assays can be performed on significant gram-negative entericand non-enteric isolates (e.g., Staphylococcus aureus, species ofbacillus, and yeast isolates.

In any of the present methods, the original sample can be aliquoted, andportions can be subjected to ancillary testing.

Identifying bacteria: Any method known in the art can be used toidentify a given bacterium in stool samples. For example, the presentmethods can include a step of identifying Clostridium difficile, andthis may be done through identification of a toxin produced by theseorganisms. C. difficile is a gram-positive, anaerobic, spore-formingbacillus that is the most commonly identified cause ofantibiotic-associated diarrheal disease. When broad-spectrum antibioticuse suppresses the normal intestinal flora, selective pressure allowstoxigenic strains of C. difficile to multiply. The toxins produced bytoxigenic C. difficile strains, toxin A and toxin B, have enterotoxicand cytotoxic effects, respectively. Disease may vary from mild diarrheato a condition known as pseudomembranous colitis (PMC), which can befatal if not treated.

C. difficile organisms may not cause symptoms in 2-3% of healthy adults,20-30% of hospitalized patients, and 50% of children under two yearsold. Thus, culture alone may be inadequate to document C. difficiledisease.

C. difficile antigens, including toxin A and toxin B, can be detectedwith specific binding proteins (e.g., antibodies). Commerciallyavailable assays, such as the ProSpecT® Clostridium difficile Toxin A/BMicroplate Assay, can be used for convenience.

Other intestinal bacteria that can be assessed include Helicobacterpylori, which may be of particular note due to its association withgastritis, gastric ulcers, and stomach cancer. Virulence factors includea vacuole-promoting cytotoxin and a potent urease enzyme, which createsan alkaline microenvironment that may allow continued growth despitestomach acidity. Typically, infected hosts mount an inflammatoryresponse that accounts for much of the tissue damage. This organism canbe harbored in a “colonized” state in which no symptoms of gastricdisease are present.

H. pylori is a small, curved, gram-negative bacillus. It is oxidase,urease, and catalase positive. It is slow-growing on culture andrequires specific conditions for growth. These features can be exploitedin testing for the presence of H. pylori in a sample. Identification canalso be made through urea-breath testing, serological tests, and withgastric biopsy tissue. Commercially available kits for detecting H.pylori include the Premier Platinum HpSA enzyme immunoassay. This is aqualitative assay for detecting H. pylori antigens in human stool. Itemploys a plurality of monoclonal anti-H. pylori capture antibodiesadsorbed onto microwells. Diluted patient samples and aperoxidase-conjugated antibody are added to the wells, and any unboundconjugated antibody is subsequently washed off. Enzyme substrate isadded to produce a colored reaction product that can be detectedvisually or spectrophotometrically.

Identification of protozoa: To assess altered gut flora, one can alsotest stool samples for protozoa. For example, fecal slides (e.g.,stained fecal slides) can be used to identify intestinal protozoancysts, ova, and trophozoites. Slide-based procedures are advantageous inthat the slides are semi-permanent; they can be preserved forre-inspection and can be shipped (e.g., to experts for consultation).While protozoa can also be detected by direct smear and concentrationtechniques, slide preparations are generally considered to be morereliable for detecting protozoa. For example, the slides can be preparedso they include areas of both thinner and thicker stool density, whichis optimal for recovery and detection of parasites.

Stool samples can be prepared in several ways. While single samples canbe tested, it may be preferable to test multiple samples (e.g., 2-4samples) that were collected on different days or to pool multiplesamples from the same patient (e.g., three samples collected over thecourse of about one week). The samples can be stored in SAF preservationmedium (sodium acetate, 10% formalin), homogenized (e.g., by vortexing)and aliquotted. One or more aliquots can then be filtered andconcentrated by centrifugation. The supernatant is discarded, and theresulting sediment is analyzed. Samples placed in SAF medium should bestable for about 60 days at room temperature.

By way of illustration, multiple stool samples from a single patient canbe concentrated by: (1) removing the caps from each patient sample; (2)pouring an aliquot from each of the patient's sample vials into a clean,appropriately labeled, empty vial and mixing thoroughly; (3) capping thesingle vial including the mixed samples; (4) suspending the sample; (5)centrifuging the sample; (6) discarding the supernatant; and (7)reconstituting the pelleted sample. This procedure can be carried outusing SpinCon® tubes (Meridian Diagnostics, Inc.) and Para-Pak Ultra®caps (Meridian Diagnostics, Inc.). In that event, the procedure can becarried out by: (1) removing the caps from each patient sample; (2)pouring an aliquot from each of the patient's sample vials into a clean,appropriately labeled, empty vial and mixing thoroughly; (3) capping thesingle vial including the mixed samples with a Para-Pak Ultra®filtration cap, removing the outer cap with the key provided, andbreaking off the tab located inside the cap; (4) placing a SpinCon® tubeonto the Para-Pak Ultra® cap, inverting and tapping the tube gently onthe counter to allow approximately 3-5 ml of specimen to filter throughthe cap into the SpinCon® tube; (5) removing the SpinCon® tube from thecap and placing a plug cap on the tube; (6) centrifuging the tube (e.g.,at about 500×g for 10 minutes); (7) removing the tub from the centrifugeand removing the top half of the tube; (8) removing half of thesupernatant with a disposable plastic pipette from the bottom half ofthe SpinCon® tube and placing it in a 13×75 mm, appropriately labeledtube for EIA procedures; (9) reconstituting the sample by vortexing andpouring roughly half of the reconstituted sample into a clearpolystyrene tube; and (10) recapping the bottom half of the SpinCon®tube and capping with a fresh plug cap. See also, Ash and Orihel,“Parasites: a guide to laboratory procedures and identification”, ASCPPress 45-7-013-00 ISBN: 0-89189-231-1, 1987; Yang and Scholten, Am. J.Clin. Path. 67:300-304, 1977; and Meridian Diagnostics, Inc., packageinserts for SpinCon and Para-Pak Ultra filtration devices, Rev. 2/01.

A suspension of a concentrated stool sample can be applied to a standardmicroscope slide along with Lugol's (5%) iodine as a contrast agent.Parasite ova and cysts are detected microscopically and identified bymorphology (e.g., shape, size, number of nuclei, cell structure, andother morphological attributes). PVA-preserved specimens are notrecommended for iodine-stained preparations, but concentrated SAF orformalin-preserved stool can be used.

By way of illustration, one can prepare a slide by: (1) inserting apipette into the tube or other vessel containing a stool sample andmixing; (2) removing a sample with the pipette and placing about onedrop onto a plain microscope slide; (3) adding about one drop of iodinesolution to the drop of sample; (4) mixing the iodine and stool samplewith the corner of a glass coverslip; and (5) placing a coverslip overthe sample. The preparation should be reasonably transparent. If it istoo thick, it can be diluted with saline before the coverslip is placed.By microscopic examination, one can detect helminthes, ova, or larvae,and amoeba. Artifacts such as white blood cells and red blood cells mayalso be noted.

Giardia cysts are typically 8-19 μm in diameter. Enterobacter (e.g., E.coli) cysts have eight or more nuclei and are 15-25 μm in diameter.Chilomastix cysts are lemon-shaped, uni-nucleate, and 6-10 μm indiameter.

For trichrome slide preparation, samples can be concentrated (e.g., bythe process described above) and prepared for staining by making a 1:1dilution of the reconstituted sample and PVA in a tube (e.g., apolystyrene tube). The sample is then poured from the tube onto a papertowel, which absorbs the PVA but not the sample. The sample can then betransferred to a microscope slide using an applicator. Moving theapplicator in a back-and-forth motion while rolling allows for a varieddistribution of material on the slide, including thicker and thinnerareas for examination. After transfer to the slide, the sample can beleft to dry at room temperature.

Batches of samples prepared for trichrome staining and examination canbe run with positive and negative controls. For example, two controlslides can be stained with each batch of patient specimens; a negativecontrol slide can be made from a sample known to be free of parasiticorganisms and a positive control slide can be made from a sample knownto contain protozoan cysts and trophozoites.

Trichrome staining is a rapid staining procedure that provides excellentdifferentiation of internal structures of intestinal parasites andseparation of the organisms from background material. It is also rapid,easy, and suitable for fecal smears. The stain itself is the Wheatleymodification of Gomori's trichrome stain for tissue sections, and it ishighly stable. With well-fixed fecal smears, the stainingcharacteristics of organisms are reasonably consistent, and the colorcontrast between organisms and background material render the organismsmore easily visible than in hematoxalin-stained smears. The cytoplasm oforganisms will usually stain a blue-green to purple, whereas nuclearchromatin, chromatoid bodies and other inclusions will stain red toreddish purple.

Suitable specimens include human stool samples in SAF preservative thathave been concentrated with PVA added. The sample can be applied tolabeled microscope slides and allowed to air dry (as described above).By way of illustration, we provide the following protocol, which can becarried out with a Jung Autostainer® instrument. The reagents requiredinclude alcohol (70% and 95%), acid alcohol (90%), and iodine alcohol(70%). Once the Jung Autostainer® has been turned on andself-initializes, it is ready to be loaded with the reagents and slides.The staining proceeds automatically as follows: (1) 70% iodine alcoholfor 5 minutes; (2) 70% alcohol for 2-3 minutes; (3) 70% alcohol for 2-3minutes; (4) trichrome stain for 10 minutes; (5) 90% acid alcohol dip;(6) 95% alcohol dip; (7) 95% alcohol dip; (8) 95% alcohol for fiveminutes; (9) 95% alcohol for five minutes; (10) 95% alcohol for fiveminutes; and (11) hemo-de solution for 25 minutes. The slides should notbe allowed to dry out before a coverslip is applied. The coverslip canbe a liquid coverslip such as Flo-Texx®.

In connection with these preparatory and staining methods, one can alsoconsult: American Society of Clinical Pathologists, LaboratoryDiagnostics of Intestinal Parasite Infection, CDC training coursematerial; Wheatley, Am. J. Clin. Pathol. 21:330-991; Ash and Orihel,supra; NCCLS, Procedures for the Recovery and Identification ofParasites from the Intestinal Tract. M28-A Vol. 17 No. 23, 1997; andJung Autostainer® Instruction Manual.

Upon inspection under the microscope, the cytoplasm of protozoantrophozoites should be a blue-green color and may be tinged with purple.Cysts tend to be more purple. Nuclei and inclusions (chromatoid bars,RBCs, and bacteria) and Charcot-Leyden crystals have a red color and aresometimes tinged with purple. Glycogen is dissolved by the fixatives andappears as a clear area in the organism. The background material usuallystains green, which provides strong contrast with the protozoa. Asnoted, the contrast is often sharper than that observed with hematoxylinstaining. Helminth ova, Balantidium coli, Entamoeba coli cysts andIsospora belli oocysts are best seen in wet preps, and acid-fast stainsare recommended for Cryptosporidium.

The protozoa, host cells, yeast cells, and any other artifacts can becounted if desired and classified as rare, few, moderate, or many. TheTable below provides recommended limits for these categories.

Wet Prep (concentration) Enumeration Protozoa (#/20 40X fields) Helminth(#/22x40 coverslip) Rare  <4  <4 Few 4-6  4-6  Moderate 6-20 6-20Many >20 >20

For yeast, 1-3 per hpf constitutes a rare number; 4-5 per high poweredfielf constitutes few; 6-10 per hpf constitutes a moderate number;and >10 per hpf constitutes many.

Other parasites that can be assessed in the context of the presentmethods include Giardia and Cryptosporidium. These organisms can bedetected in a variety of ways, including enzyme immunoassay (EIA) forspecific antigens (e.g., GSA 65 and CSA) present in aqueous extracts ofhuman stool samples. More specifically, one can employ the ProspecT®microplate assay according to the manufacturer's instructions tosimultaneously detect GSA 65 and CSA. Briefly, stool specimens are addedto break-away microplate wells to which anti-GSA 65 and anti-CSAantibody are bound. The antibodies are conjugated to an enzyme thatlater produces a detectable product when bound by an antigen. Afterunbound material is washed away, an enzyme substrate (TMB where theenzyme is horseradish peroxidase) is added to the well. In a positivereaction, the development of color indicates that parasitic antigens arepresent in the sample. In a negative reaction, antigen is either absentor present in insufficient amounts to be detected, and no coloredreaction product develops. Acceptable samples include unpreserved stool,stool samples in Cary-Blair medium, SAF medium, or 10% formalin.Ideally, fresh samples have been frozen, samples in Cary-Blair mediumare refrigerated (2-8° C.) and tested within one week of collection, andsamples in SAF, 10% formalin or MF transport media are refrigerated orstored at room temperature and tested within two months. Stool samplestreated with PVA are not acceptable for testing.

Similar assays can be configured to detect other antigens so long as anantigen-specific binding protein (e.g., an antibody) is available. Forexample, the ProspecT® Entamoeba histolytica/dispar microplate assay isavailable for the detection of EHSA (E. histolytica specific antigen).

In assays such as the ProspecT® assay, acceptable samples includeunpreserved stool, stool samples in Cary-Blair medium, SAF medium, or10% formalin.

Any of the methods described herein can include a step of identifying apatient who is a candidate for evaluation. In some cases, a physician orother healthcare provider may suspect giardiasis, caused by Giardialamblia, or cryptosporidiosis, which is caused by Cryptosporidium sp. G.lamblia are transmitted via ingestion of viable cysts, and this occursmore frequently among children and in groups of people who live in closequarters. The acute stage occurs about 12-20 days after ingestion andtypically lasts only a few days. The patient may complain of diarrheaand flatulence without the presence of blood in the stool or otherhallmarks of inflammatory bowel disease. Malaise, malabsorption,anorexia, abdominal cramping, weight loss, and general weakness are someof the other manifestations of infection. The disease can linger forweeks or months as a chronic presentation.

Cryptosporidiosis is a serious intestinal disease that is commonlyobserved in people who work with young children, animal handlers, andtravelers, and it is particularly dangerous to individuals who areimmunosuppressed or otherwise weakened or compromised. Symptoms includediarrhea, abdominal pain, nausea and vomiting, fever, malaise andrespiratory problems, which may last from several days to more than amonth.

Any of the methods described herein can include a step in which theresults of one or more of the assays described herein are correlatedwith patient symptoms and any other clinically relevant information.

EXAMPLES Example 1

A 32 year-old male patient presents with abdominal discomfort andalterations in bowel movements (3-4 times/month) since returning fromtraveling in Nepal six months earlier. He has been evaluated forparasites at a local hospital. An EIA for Entamoeba histolytica wasnegative. He has difficulty when eating sweets or other sugar-richfoods, which leads to an urge to use the bathroom. The patient presentedto a doctor who suspected Irritable Bowel Syndrome. While the doctor wasconcerned about the potential underlying causes of pain and discomfort,hospitalization and colonoscopy were not considered necessary at thattime. The doctor elected to perform stool testing to evaluate digestion,inflammation, infection, and gut microflora. The following results werenoted:

-   Pancreatic Elastase=188 (nl>200)-   Calprotectin=22 (nl<50)-   Eosinophilic Protein X=8.9 (nl<7.0)-   Microscopy—positive for Blastocystis hominis

The patient was prescribed pancreatic enzymes with meals, treated with aprescription anti-parasitic agent, and offered L-glutamine to supporthealing of the gastrointestinal lining. Within one week, the patientbegan to experience improvement in symptoms he had experienced for theprevious six months. L-Glutamine was continued for six weeks, andpancreatic enzymes were continued for three months, as were supplementalprobiotics.

After three months, the symptoms had completely resolved and medicationscould be ceased. The patient remains symptom-free more than one yearfrom the initial visit.

Example 2

A 42 year old female presents with a three year history of irregularbowel movements and intermittent lower abdominal pain, for which she hasseen her previous primary care provider many times. She also has beendiagnosed with depression and is currently using an SSRI for depression.She notes that the tri-cyclic anti-depressant she used previously didnot significantly affect her abdominal pain. She has used fiber,anti-diarrheals, probiotics, and dietary changes in the past, to noavail.

Upon presentation the doctor chooses to run a stool test to evaluate theroot cause of this illness. While waiting for the stool test to return,empiric use of probiotics at 10 billion cfu per day has made nodifference.

Upon testing, pancreatic elastase was determined to be 482 (nl>200). Atthis level, one would conclude that the patient has normal pancreaticfunction and no need for pancreatic enzyme support. Calprotectin wasdetermined to be 286 (nl<50). This level of elevation indicates severegastrointestinal tract inflammation and warrants referral to aspecialist for further investigation (i.e., colonoscopy). There was noevidence of parasite infection or altered gut flora.

While no clinical intervention is indicated, the patient was referredfor colonoscopy for inflammatory changes in the gastrointestinal tract.She is diagnosed with ulcerative colitis and placed on steroids and5-ASA. About 1-10% of patients with IBS go on to develop IBD. The stooltest, with its use of a stool-based marker for GI inflammation, allowedfor early detection and appropriate referral of this patient to thegastroenterologist. Subsequent evaluations of GI inflammation with thecalprotectin biomarker can be used to monitor mucosal healing anddetermine if repair is sufficient or complete.

Example 3

A 21 year old female presents with a history of intermittent recurrentabdominal cramping, bloating/gas and occasional diarrhea over the pasttwo years. She has been evaluated by her college health clinic onnumerous occasions, was told that her symptoms were primarily ‘stressrelated,’ and was advised to cut back on her coffee intake during theday. She was recently evaluated for complaints of fatigue, and wasdiagnosed with hypothyroidism (including elevated thyroid antibodies)for which she is being treated. While home during a break, the patientwas asked to do a stool analysis by her primary care doctor who wasconcerned that some underlying etiology had not been determined. Resultswere significant for the following:

-   Pancreatic Elastase=150 (nl>200)    -   At this level, the pancreatic elastase clinically indicates        moderate pancreatic insufficiency and need for pancreatic enzyme        support. Evaluation of the underlying cause of pancreatic        insufficiency is warranted.-   Calprotectin=22 (nl<50)    -   A normal Calprotectin level indicates no inflammation due to        neutrophilic activity in the gastrointestinal tract.-   Eosinophil Protein X=13.1 (nl<7.0)    -   An elevated Eosinophil Protein X may be associated clinically        with Celiac disease, parasitic infections, and/or IgE mediated        food allergies.-   Microscopy—no evidence of parasite infection or altered gut flora.

Given that Celiac disease can be consistent with the patient's clinicalsymptoms, and may be indicated on stool analysis by an elevatedEosinophil Protein X and compromised pancreatic exocrine function (lowPancreatic Elastase), the doctor decided to evaluate the patient forCeliac disease. Blood serologies (positive Tissue Transglutaminase IgAand positive anti-Endomysial antibody) confirmed the diagnosis of Celiacdisease. The patient was placed on a gluten-free diet and givenpancreatic digestive enzyme support. At follow-up, the patient reportedthat her symptoms had markedly improved—appearing only during times ofdietary indiscretion.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of assessing a patient'sgastrointestinal health, the method comprising: (a) providing a stoolsample from the patient; and (b) subjecting the sample to three tests: afirst test for maldigestion, a second test for inflammation, and a thirdtest for gut flora. 2-23. (canceled)